Structural study of hydra nematocyst wall assembly

The present thesis includes four parts. All of them are devoted to the
investigation of the nematocyst capsule wall structure and assembly. The nematocyst
wall is unique in its resistance to high pressures accomplished by a pronounced
elasticity. The capsule develops inside a Golgi vesicle using the vesicular membrane
as a substrate. Recent studies demonstrated that the mature nematocyst capsule
wall is a compact structure of several closely arranged layers of globular building
units termed “capsulomers”. These globules have slightly heterogeneous size
distribution of 20 nm average diameter. Capsulomers were shown to be
heterooligomers of NOWA and minicollagens which are crosslinked by disulfide
bonds. In addition capsulomer precursors composed of NOWA alone were identified
at the certain step of the nematocyst capsule development.
Minicollagens and NOWA share similar cysteine rich domain that is presumably
responsible for the disulfide dependent capsulomer formation and further covalent
cross-linking of the capsulomers. However exact mechanism of the highly ordered
intra- to inter- disulfide isomerization leading to the formation of the capsule wall
remains unclear. The structure of the common cysteine rich domain was not known
also.
(Chapter 1) In order to investigate whether NOWA alone assembles to
capsulomer-like structures the full-length protein was expressed in mammalian cells,
purified and investigated by various techniques. Mammalian expression system was
chosen to provide post-translational modifications of the protein and correct disulfide
connectivity. Monomeric and oligomeric fractions of the recombinantly expressed
protein were isolated and studied by transmission electron microscopy. NOWA
monomers revealed compact globular structures. Oligomeric fraction of protein was
found to be self-assembled capsulomer- like globules of the average diameter similar
to that of native wall capsulomers. The capsulomer-like structures have melted upon
reduction. Similar experiments were performed with the recombinantly expressed
NOWA fragment containing eight Cys-rich domains (ONCRD). This construct was
shown to assemble into ring-like structures rather than spherical particles. Small ringlike
structures were further associated in chains of different curvature. Thus assembly
of the recombinant NOWA to capsulomer-like structures was concluded to be a
feature of its cysteine rich domain although other protein domains needed to provide a control of disulfide isomerization. Furthermore capsulomer-like structures exhibited
calcium dependent clusterization. This property of the recombinant protein was
discussed to explain natural behavior of the capsulomer precursors during
nematocyst development.
(Chapter 2) Nematocyst wall maintains close contact with the membrane of the
Golgi vesicle during capsule development as well as in mature form. To investigate
whether NOWA is able to interact with the membrane surfaces recombinantly
expressed protein was studied by several techniques. NOWA binding to the model
lipid monolayers and bilayers was directly visualized by electron microscopy. Surface
plasmon resonance kinetic studies revealed high affinity of the capsulomer-like
structures to the negatively charged surface of lipid bilayer (KD~ 100 nM). Calcium
induced clusters of the NOWA capsulomers exhibited ten times lower membrane
affinity. This provides an explanation for in vivo dissociation of the capsulomer
precursor clusters. Two parts of NOWA involved in the membrane binding were
identified: C-type lectin domain and basic C-terminal sequence. The cysteine rich
domain of the protein had shown no membrane affinity.
(Chapter 3) The minicollagen and NOWA cysteine-rich domains (MCRDs and
NCRDs respectively) are believed to function in a switch of the disulfide connectivity
from intra- to intermolecular bonds during maturation of the capsule wall. The N- and
the C-terminal MCRDs as well as 8 cysteine rich domains of NOWA are homologous
and share the cysteine pattern CXXX(X3)CXXXCXPXCXXXCC. The peptide
comprising the last 24 residues of the minicollagen-1 was produced synthetically and
successfully refolded by oxidation under low protein concentration. The solution
structure of the C-terminal MCRD was determined by 1H NMR technique. Disulfide
connections Cys2-Cys18, Cys6-Cys14, Cys10-Cys19 were found that constrained
the structure into a compact new fold.
(Chapter 4) The solution structure of the first cysteine rich domain of Hydra
nematocyst wall protein NOWA (NCRD1) has been determined using homonuclear
and heteronuclear NMR techniques at natural abundance. The elucidated peptide
composing 25 amino acids was produced synthetically and oxidized. The NCRD1
has revealed the disulfide pattern identical to that of the MCRD. Moreover NOWA
domain exhibited overall structure topology similar to the topology of minicollagen
domain structure. Despite the differences in the N-terminal structures two peptides revealed the same structural fold defined by conserved cysteines connection and ßturns.
The MCRD and NCRD1 both have single tyrosine in their sequences. Tyrosine
fluorescence quenching by disulfide bonds was observed in both peptides. The
oxidative refolding of MCRD and NCRD1 has been monitored by tyrosine
fluorescence. Resulting kinetics were fitted with mono-exponentials suggesting a
simple kinetic mechanism that arises from the quenching by only one particular
disulfide bond in each case. The Cys2-Cys18 bond in case of MCRD and Cys6-
Cys14 in case of NCRD1 are discussed to be responsible for fluorescence quenching
effect. Oxidative refolding rate constants related to the formation of indicated bonds
were determined.